The clathrin coated vesicle[unreadable] cycle drives synaptic vesicle recycling and is essential for the maintenance of[unreadable] repeated rounds of neurotransmission. Many of the structural components of the[unreadable] clathrin endocytic machinery have been identified, but our understanding of the[unreadable] molecular mechanisms underlying and regulating clathrin-mediated endocytosis is[unreadable] still in its infancy. Moreover, only one enzyme, the GTPase dynamin, has been[unreadable] identified to play a role in clathrin-mediated endocytosis even though these[unreadable] reactions are ATP-dependent. My laboratory has pioneered the development of a[unreadable] set of cell-free assays that together, faithfully and efficiently reconstitute[unreadable] each step in the clathrin-coated vesicle cycle, including coated pit assembly,[unreadable] coated vesicle formation and the sequential uncoating reactions that recycle[unreadable] the coat proteins, freeing the enclosed vesicles. Using these[unreadable] well-characterized assays, we are in a unique position to elucidate the[unreadable] molecular mechanisms that govern synaptic vesicle recycling. The long-term goal[unreadable] of this proposal is to identify the minimal set of cytosolic and peripheral[unreadable] membrane proteins required to reconstitute clathrin-mediated endocytosis and to[unreadable] complete the clathrin-coated vesicle cycle. To meet this objective, we propose[unreadable] the following Specific Aims: 1) To develop a novel assay for coated vesicle[unreadable] budding from highly enriched plasma membranes to enable identification of[unreadable] peripheral and integral membrane components of the endocytic machinery; 2) To[unreadable] determine the hierarchy of events leading to coated pit assembly and endocytic[unreadable] clathrin coated vesicle formation; 3) To identify minimal cytosolic components[unreadable] required for endocytic coated vesicle formation; 4) To identify proteins that[unreadable] regulate the uncoating reaction and to test the role of hsc70, the uncoating[unreadable] ATPase in vivo; and, 5) To develop transient expression systems in primary[unreadable] neurons to demonstrate that protein interactions and mechanisms identified in[unreadable] vitro are required for synaptic vesicle recycling in vivo.